Our long-term objectives are to understand the mechanisms of DNA evolution and to elucidate the evolutionary history of genes and organisms. The specific aims for the next five years are to study: 1. Methods for estimating the number of nucleotide substitutions under nonequilibrium conditions. 2. Methods of tree reconstruction. a. Statistical tests. New tests will be developed. Within population variation effects will be incorporated. The rigorousness and efficiency of the bootstrap approach will be examined. b. Tree reconstruction under unequal rates of evolution. A method suitable for inferring deep branchings in the living world, e.g., the origin of eukaryotes, will be developed. The reliability of outgroups will be studied. 3. Deletions and insertions in sequence evolution: their frequency and location of occurrence, the size of gap events and the relative frequencies of spontaneous deletion and insertion. These questions will be studied in pseudogenes, introns, spacer regions and coding regions. The hypothesis that spontaneous deletion occurs more often than spontaneous insertion will be tested. 4. Population genetics of short tandem repeats and the rate and pattern of mutation to new length alleles. 5. Evolution of cellular fatty acid- and retinol-binding proteins and related proteins. A phylogenetic tree will be constructed for members of this superfamily, and used to infer how different structural domains have been modified in evolution. One interesting question is whether heart fatty acid-binding protein (FABP) is more distantly related to liver and intestine FABPs than to other members of the superfamily. The rate of evolution will be calculated and used to infer the stringency of structural requirements in each domain of the proteins. The hypothesis that serum retinol binding protein is related to members of this superfamily will be tested. 6. The origin, age, and phylogeny of eukaryotes. The origin of the eukaryotic nucleus and the date of the eukaryote-prokaryote divergence will be inferred. The phylogeny of eukaryotic microorganisms will be reconstructed with particular interests in the earliest branching in eukaryotes, the date of this branching, and the evolutionary position of the ciliates and the dinoflagellates.